Pharmaceutical Confirmation Using and Nuclear Magnetic Resonance Proof of Structure – Clindamycin Application Note

Pharmaceuticals

Authors Abstract

Dave Russell and Tiffany Payne This note investigates the structure of clindamycin in an integrated fashion using an Agilent Technologies, Inc. Agilent 500 Ion Trap LC/MS and an Agilent 400-MR NMR spectrometer. The data 5301 Stevens Creek Boulevard allows for the rapid confirmation of the . Santa Clara, CA 95051 USA Introduction One of the basic spectroscopic tasks widely performed in the pharmaceutical indus- try is confirmation of structure for a known compound. This is a critically important function with relevance in many areas, including medicinal , process research, pharmaceutical development, manufacturing, and numerous regulatory operations.

Two of the key spectroscopic techniques used to accomplish this task are mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). These methods provide a wealth of complementary structural information. Mass spectrome- try is used to determine the molecular formula for a given compound and can provide structural information through MSn analysis. Nuclear magnetic resonance spec- troscopy is used to determine the type and connectivity of individual within a structural framework. When used in an integrated fashion, these techniques can unambiguously establish the chemical identity of a pharmaceutical compound.

Clindamycin is a widely prescribed lincosamide antibiotic that acts through inhibi- tion of bacterial synthesis. This active pharmaceutical ingredient is a semi- synthetic material derived from lincomycin, a isolated from the acti- nobacterium S. lincolnensis. Careful characterization of the bulk drug is important as the synthetic process is known to result in impurities such as clindamycin B, 7-epiclindamycin, and unreacted lincomycin. As an example of a typical confirmation of structure analysis, we have investigated the structure of clindamycin in an inte- 9 Cl O H 8 O 19 grated fashion using an Agilent 500 Ion Trap LC/MS and an H O 5 7 11 N Agilent 400-MR NMR spectrometer. 4 6 12 N 15 Cl 3 13 H O O H 14 O H O 2 HO N 16 17 N S 20 O H 18 HO

SCH3

Figure 1. Clindamycin.

Results and Discussion

Primary mass spectral investigations were performed using LC/MS analysis. The MS data were collected with an Agilent 500 Ion Trap LC/MS using TurboDDS data-dependant scan- ning software. This software allows for collection of a full scan “survey” as well as MSn data for structural analysis. As shown in Figure 2, the analyte displayed a signal representing the protonated at m/z 425.2, consistent with the expected molecular formula of C18H35ClN2O5S.

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Figure 3. Proton NMR spectrum of clindamycin. These data can be inter- preted to provide information about the number of 1H atoms in a given chemical environment and structural relationship between those atoms.

Clindamycin displays several interesting chemical motifs. The presence of the substituted pyrrolidine ring is revealed by the observation of an m/z 126 ion in the full scan mass spectrum of clindamycin. The NMR data also confirm this assignment and show the presence of a contiguous seven-spin system (for example, H12 through H18). The Heteronuclear Single Quantum Coherence (HSQC) data set allows identification of

Aquired range m/z the two methine, four methylene, and one methyl resonances Figure 2. Full scan spectrum of clindamycin. expected for this carbon chain, while the responses observed in the Correlated Spectroscopy (COSY) spectrum establish The molecule is also well behaved magnetically and displays molecular connectivity between the heteronuclear spin pairs. NMR spectra that are nearly first-order, even at the modest The chemical shifts observed for H/C12 and the strongly 9.4 Tesla field of a 400-MR spectrometer (Figure 3). anisochronous H/C15 pair are consistent with their attach- ment to the cyclic pyrrolidine . Finally, the H/C19 methyl group yields a three-bond HMBC response to both C12 and C15, thereby completing this structural fragment.

2 The second major structural fragment in this molecule is the F1 (ppm) thiosaccharide ring with its pendant chloroethyl tail. The observation of fragment ions at m/z 389 and m/z 377 are 1.5 indicative of the loss of HCl and HSCH3, respectively. A frag- 2.0 ment ion was observed at m/z 407, corresponding to the loss of water from the protonated molecule. This m/z 407 ion 2.5 loses a second molecule of water to yield an m/z 389 ion that 3.0 H6→ H4 is distinct from the previously observed facile loss of neutral 3.5 HCl from the [M+H]+ ion. The process by which these ions are generated can be differentiated by independent observa- 4.0 35 37 tion of the fragmentation pathway for the Cl and Cl iso- 4.5 topes of the parent compound. In a similar fashion, a fragment H4 →Η6 5.0 ion is observed at m/z 335, consistent with the neutral loss of 2-(methylsulfanyl) ethanol from the thiosaccharide ring 5.5 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 (Figure 4). F2 (ppm) Figure 5. Two-dimensional NOESY spectrum of clindamycin. Dipolar relax- ation data such as these provide information about the spacial relationships between proton atoms. The H4 to H6 1,3-cis-diaxial interaction is labeled.

Finally, connectivity between the two halves of the molecule is provided by responses in the Heteronuclear Multiple Bond Correlation (HMBC) data set. The H7 and H13 resonances are both observed to respond with a carbon resonance at 167.9 ppm. This is the chemical shift expected for the only quaternary carbon found in clindamycin, the C11 carbonyl atom.

Cl OH O HO N N Figure 4. MS/MS spectrum of clindamycin. Observation of fragment ions such as m/z 335 provide structurally significant information about O H the atomic structure of the precursor ions. HO S The 1H NMR spectrum of clindamycin displays the cluster of methine resonances between 3.5 and 5.3 ppm expected for a sugar moiety. The COSY and HSQC data sets, when used in Figure 6. Structurally significant three-bond HMBC responses (H & C) observed for clindamycin. conjunction, allow unambiguous assignment of the contigu- ous eight-carbon spin system beginning with H/C2 and extending through to the terminal H/C9 methyl resonance. The large 11.1 Hz coupling constant observed between H3 and H4 indicates the expected 1,2-trans-diaxial relation- ship of these two proton atoms. All the other coupling con- stants in the carbohydrate ring are more modest (for exam- ple, ~ 4.2-5.9 Hz), as predicted based on the stereochemistry of the molecule. Further confirmation of the relative stereo- chemistry in the carbohydrate ring is provided by the 500 ms 2-D Nuclear Overhauser Enhancement Spectroscopy (NOESY) experiment. As shown in Figure 5, H4 and H6 dis- play reciprocal dipolar relaxation as predicted based on their 1,3-cis-diaxial relationship.

3 Conclusion

When used in an interdisciplinary fashion, modern spectro- scopic techniques provide extremely powerful tools for the investigation of chemical . The integration of NMR and MS data interpretation allows for the rapid confirmation of chemical structure with very high confidence in the result. As shown here using clindamycin as a example, the struc- tural data required to document a characterization report were acquired in an automated fashion using an Agilent 400-MR NMR spectrometer and Agilent 500 Ion Trap LC/MS.

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© Agilent Technologies, Inc., 2011 Printed in the USA February 24, 2011 SI-1861